1. Field of the Invention
This invention relates to compositions containing 5.alpha.-dihydro-19-norethisterone (5.alpha.-DHNET) and its acyl derivatives as in vivo inhibitors of the enzyme aromatase. As a result of this enzyme inhibitory effect, the present compositions can be used to prevent and treat endocrine dependent conditions such as gynocomastia, and estrogen-dependent breast or uterine cancers.
2. Discussion of the Background
Aromatase is an enzyme which catalyzes the conversion of androgen into estrogen, the terminal aromatization step of estrogen biosynthesis. It is a membrane-bound complex system, which catalyzes a series of reaction steps with multiple mono-oxygenations. Because of the complexity of the system, the precise pathway or reaction mechanism of aromatization has not yet been established, nor has the isolation of pure enzyme been achieved. At least two distinct forms of human placental aromatase are known. See Osawa and Higashiyama, in Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Coon, M. J. et al.), volume 1, pp. 225-228, Academic Press, London and New York (1980).
The product of aromatase action, estrogen, is not only essential for reproduction and development but also promotes the growth of estrogen dependent cancers.
Approximately one third of the cases of human breast cancers require the female hormone estrogen for their growth and regress when the tumors are deprived of the hormone. Removal of the source of estrogen is an effective method of treating breast cancers, and other endocrine-dependent cancers including uterine cancer.
The ovary is the major source of estrogen in premenopausal women and oophorectomy (excision of the ovary) is the classical treatment of premenopausal patients with advanced breast cancer. In postmenopausal patients, the sites of estrogen biosynthesis are peripheral tissues such as fat, skin, muscle, and the tumor itself, where the conversion of androgen to estrogen is catalyzed by aromatase. These peripheral tissues are not subject to excision by surgical methods, but their aromatase activity may be chemically inhibited by use of aromatase inhibitors. Several agents, such as aminoglutethimide, testololactone, and 4-hydroxyandrostenedione, which have been used to successfully treat breast cancer, are aromatase inhibitors. However, these drugs are not particularly specific or highly potent, and they have deleterious side effects. There is, therefore, a need to develop better agents with fewer side effects for long term treatment to prevent the onset and growth of endocrine-dependent tumors. In this respect, 5.alpha.-DHNET was found to be particularly valuable by in vivo pharmacological studies.
Another condition associated with an imbalance in the amount of estrogen is known as gynecomastia. This is a pathological condition resulting in enlargement of the male breast. In gynecomastia, due either to normal or abnormal causes the enlargement is believed to result from disturbance of the normal ratio of active androgen to estrogen in plasma or within the breast itself. In men given diethylstilbestrol, histological changes in the male breast resemble those in other forms of clinical gynecomastia, a finding in keeping with the concept that gynecomastia is the result of an estrogen effect. Estradiol formation in the normal man occurs principally by the conversion of circulating androgens to estrogens in peripheral tissues. Feminization results when there is a significant decrease in the ratio of production of testosterone to estradiol, and this may be a result of diminished testosterone production or action, enhanced estrogen formation, or both processes occurring simultaneously. Inhibitors of aromatase would have an inhibitory effect on gynecomastia when the condition is associated with enhanced estrogen formation in the male.
5.alpha.-DHNET is a reduction product of the contraceptive progestogen, norethisterone. The structure of this compound is as follows: ##STR2## wherein R.sub.1 is hydrogen.
It is a known compound which has been synthesized as an intermediate from norethisterone by reduction with lithium in liquid ammonia (A. Bowers, H. J. Ringold and E. Denot, J. Am. Chem. Soc. 80, 6115-6118 (1958)). Bowers et al reported no utility whatsoever for this compound. Unlike norethisterone, 5.alpha.-DHNET is not aromatizable by aromatase due to the saturation of the 4-double bond and thus it does not possess the adverse consequence of being converted to an estrogenic substance after being given to the patient. This compound is the major reduced metabolite in humans of norethisterone which has been widely administered on a long term basis to women of reproductive age for the past thirty years as the major ingredient of the contraceptive pill. Thus, it has been shown that the compound is safe for treatment of human subjects even in the long term.
A number of substrate analogs have been evaluated as competitive inhibitors of aromatase in vitro (see Schwarzel et al., Endocrinology (Baltimore) 92, 866-880 (1973)). 4-Hydroxyandrostenedione has been found to cause regression of estrogen-dependent breast cancers of rats (Brody et al., Endrocrinology (Baltimore) 100, 1684-1695 (1977)).
Generally, irreversible inhibitors are expected to be more effective for use in vivo if they are targeted selectively toward the enzyme. Mechanism-based or suicide inactivators are designed to achieve a high degree of selectivity of irreversible inhibition through a covalent-bond formation at the active site of the enzyme, but before such compounds are acted upon by the target enzyme they are relatively unreactive and are therefore not likely to form covalent bonds with other cellular components indiscriminantly. In fact, they carry a latent reactive functional group which is transformed through normal catalytic activity of the target enzyme into the reactive species. This activation occurs after formation of the enzyme-inhibitor complex and therefore the activated inhibitor has a better chance to make a covalent bond connection to a reactive group of the enzyme at or near the active site resulting in selective inactivation of the enzyme. The compound norethisterone (17.alpha.-ethynyl19-nortestosterone) is a suicide inactivator of aromatase. 5.alpha.-DHNET has now been discovered to be an in vivo inhibitor of aromatase and it is suspected that such inhibition occurs through a mechanism based reaction.
5.alpha.-DHNET was previously found to be a very weak in vitro aromatase inhibitor (Y. Osawa, Y. Osawa, C. Yarborough & L. Borzynski, Biochem Soc. Transactions, 656-659 (1983)), requiring a 50 .mu.M concentration for a partial inhibition of human placental aromatase in vitro. This compares to 0.0082 .mu.M and 0.056 .mu.M for 6.alpha.- and 6.beta.-bromoandrostenedione by the in vitro assay with human placental aromatase (S. J. Santner, H. Rosen, Y. Osawa & R. J. Santen, J. Steroid Biochem. 20, 1239-1242 (1984)) and indicates that 5.alpha.-DHNE is a thousand-fold less potent aromatase inhibitor in in vitro assays.
The potent in vivo aromatase inhibitory activity of 5.alpha.-DHNET and suppression of the growth and incidence of breast carcinomas in experimental animals as shown in the following Examples were totally unexpected and could not be deduced by inference from the previous in vitro studies. Indeed, the in vitro studies showed 5.alpha.-DHNET to be essentially inactive; certainly not a candidate for in vivo activity. Thus, the properties of 5.alpha.-DHNET, which make it useful as an inhibitor of aromatase in vivo and as a drug for treatment and suppression of the incidence of certain endocrine-dependent cancers, were found for the first time by this invention. In general, although a variety of compounds have been shown to be in vitro aromatase inhibitors and have therefore been considered potential candidates for in vivo activity, it can now be seen that in vitro activity provides no assurance of in vivo activity.